We study the mechanism and regulation of eukaryotic protein synthesis focusing on the roles of GTPases and a family of stress-responsive protein kinases. In the first step of protein synthesis translation initiation factors promote the assembly of an 80S ribosome at the AUG codon of an mRNA. The factor eIF2 is a GTPase that facilitates binding of the specific initiator methionyl-tRNA (Met-tRNA) to the small ribosomal subunit. Base-pairing of the anticodon on the Met-tRNA with an AUG codon on an mRNA triggers GTP hydrolysis by eIF2, and release of the factor from the ribosome. The factor eIF5B is a ribosome-dependent GTPase that promotes subunit joining in the final step of translation initiation. The eIF5B is an ortholog of the prokaryotic translation factor IF2. Comparison of the X-ray structures of active GTP-bound and inactive GDP-bound eIF5B revealed lever-type domain rearrangements in the factor accompanying GTP hydrolysis. Changing the nucleotide specificity of eIF5B from GTP to XTP resulted in the requirement for both nucleotides for efficient subunit joining and peptide synthesis. Thus, in contrast to the single GTP requirement for translation initiation in bacteria, two GTP molecules are consumed during eukaryotic translation initiation. Consistent with the biochemically-defined role for eIF5B in subunit joining, yeast strains lacking eIF5B showed increased levels of leaking scanning. We found that GTP-binding to eIF5B, but not eIF5B GTPase activity, was essential for subunit joining. Mutation of the universally conserved Switch 1 motif in the eIF5B GTP-binding domain impaired cell growth and eIF5B GTPase activity, but not subunit joining. Intragenic suppressor mutations of this Switch I mutant restored near wild-type growth, but did not restore the GTPase activity of the factor. These suppressor mutations, which map to the ribosome-binding face of the factor, lowered the ribosome-binding affinity of eIF5B. We propose that GTP-bound eIF5B binds to 40S ribosome preinitiation complexes, where it stabilizes binding of the initiator Met-tRNA to the ribosomal P site and promotes subunit joining. Joining of the 60S ribosomal subunit triggers GTP hydrolysis by eIF5B, and coverts the factor into a form with low ribosome binding affinity. Thus, eIF5B is a regulatory GTPase in which GTP versus GDP binding governs the ribosomal affinity of the factor. Four protein kinases PKR, GCN2, HRI and PERK regulate translation by phosphorylating serine-51 on the alpha subunit of eIF2, converting eIF2 from a substrate to a competitive inhibitor of its guanine nucleotide exchange factor eIF2B. The kinase PKR contributes to anti-viral defense in mammalian cells, and we established a heterologous system in yeast to study PKR. High-level expression of PKR inhibits yeast cell growth, and co-expression of the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR, alleviates PKR toxicity in yeast. The M156R protein from myxoma virus is a homolog of the K3L protein. Whereas the K3L protein inhibits PKR kinase activity, we found that PKR efficiently phosphorylates the M156R protein. The M156R protein competed with eIF2alpha for phosphorylation by PKR in vitro, suggesting a possible mechanism by which myxoma virus prevents PKR phosphorylation of eIF2alpha. Twelve single amino acid changes were identified in the PKR kinase domain that restored PKR toxicity in yeast co-expressing the K3L protein. We propose that these mutations, located in the PKR kinase domain, lower the affinity of PKR for its pseudosubstrate without severely impairing substrate binding and phosphorylation. N- and C-terminal truncation analyses revealed that residues 1-180 of eIF2alpha represent the minimal substrate for efficient phosphorylation of serine-51 by PKR or GCN2. Mutations were isolated in eIF2alpha residues 49, 50, and 79-83 that impaired phosphorylation of serine-51 by GCN2 and PKR both in vivo and in vitro. Strikingly, substitution of alanine for aspartic acid-83, 32 residues from the site of phosphorylation, completely blocked phosphorylation. We propose that the eIF2alpha kinases recognize their substrate utilizing residues both nearby and remote from the phosphorylation site. The identification of a second set of mutations in residues 49, 50 and 79-83 that block translational regulation, and presumably eIF2B inhibition, but not serine-51 phosphorylation indicates that the eIF2alpha kinases and eIF2B interact with overlapping surfaces on eIF2alpha.